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Reduced Transplacental Transfer of a Subset of Epstein-Barr Virus-Specific Antibodies to Neonates of Mothers Infected with Plasmodiumfalciparum Malaria during Pregnancy

Sidney Ogolla,a,b Ibrahim I. Daud,a,c Amolo S. Asito,a,d Odada P. Sumba,a Collins Ouma,b* John Vulule,a Jaap M. Middeldorp,e

Arlene E. Dent,f Saurabh Mehta,g Rosemary Rochfordh*

Center for Global Health Research, Kenya Medical Research Institute, Kisumu, Kenyaa; Maseno University, Kisumu, Kenyab; Jomo Kenyatta University of Agriculture andTechnology, Nairobi, Kenyac; School of Physical and Biological Sciences, Jaramogi Oginga Odinga University of Science and Technology, Bondo, Kenyad; Department ofPathology, VU University Medical School, Amsterdam, The Netherlandse; Case Western Reserve University, Cleveland, Ohio, USAf; Division of Nutritional Sciences, CornellUniversity, Ithaca, New York, USAg; Department of Microbiology and Immunology, State University of New York Upstate Medical University, Syracuse, New York, USAh

Over 35% of children in a region of malaria endemicity are infected with Epstein-Barr virus (EBV) by 6 months of age. This sus-ceptibility may be linked to impaired transplacental transfer of antibodies. In this study, we determined the effect of malaria ex-posure during pregnancy on the transfer of EBV-specific maternal antibodies in a region of western Kenya that experiences en-demic malaria. Pregnant mothers were recruited and followed up until delivery to determine levels of neonatal malariaexposure. Levels of EBV lytic (viral capsid antigen [VCA], Z transcriptional activator [Zta], and early diffuse antigen complex[EAd]) and EBV latent (EBV nuclear antigen-1 (EBNA1]) and tetanus-specific IgG antibodies were measured in 70 paired mater-nal and cord blood samples using a Luminex-bead-based assay. A high proportion (63%) of the infants were exposed to malariain utero. Levels of EBV- and tetanus-specific antibodies were similar in malaria-infected mothers and in mothers who had nodetectable malaria infection. Malaria-exposed neonates had significantly lower levels of anti-EBNA1, anti-Zta, and anti-EAd an-tibodies than were seen in their mothers. In utero malaria exposure resulted in significant reductions in transplacental transferof anti-VCA-p18 and anti-EBNA1 antibodies of 13% and 22%, respectively. Neonates received significantly low levels of anti-Ztaand anti-EAd antibodies irrespective of malaria exposure levels. In multivariate analysis, in utero malaria exposure was associ-ated with a significant reduction in the transfer of anti-VCA-p18 and anti-EBNA1 antibodies to the neonates (P 0.0234 andP 0.0017, respectively). Malaria during pregnancy results in differential levels of transfer of EBV-specific antibodies from themother to the fetus. The impaired transplacental transfer of some antibodies may lead to the malaria-exposed neonates beingsusceptible to early EBV infection.

Endemic Burkitts lymphoma (eBL) is a distinct form of non-Hodgkins lymphoma and is the most common pediatric ma-lignancy in regions of malaria endemicity of sub-Saharan Africa(1). Both infection with Epstein-Barr virus (EBV) and repeatedepisodes of Plasmodium falciparum malaria are known risk factorsfor eBL (2), but the mechanism(s) by which these two agents in-teract to promote the emergence of malignant B cell clones has notbeen elucidated. Recently, we found that infants from a region ofmalaria endemicity of western Kenya were infected with EBV by 6months of age (3). Living in regions of malaria endemicity was apredictor of this early age of primary infection. This aberrant pri-mary EBV infection may set the stage for lymphoma development,as previously hypothesized (46).

The lytic and latent phases of the EBV life cycle induce distinctantibodies in response to specific lytic and latent antigens. Anti-EBV nuclear antigen-1 (EBNA1) antibodies are produced againstEBNA1, the only antigen expressed in latently infected memory Bcells and in eBL tumors (7). Anti-viral capsid antigen (anti-VCA),anti-early antigen (anti-EA), and anti-immediate-early protein(anti-Zta) antibodies are produced against their respective targetlytic antigens (8). Clinically, elevated levels of anti-VCA and anti-EBNA1 immunoglobulin G (IgG) antibodies have been used asevidence of past infection (9), while the presence of IgG antibodiesto the EBV early antigens (EAd and Zta) generally reflects recentor reactivated infections (10, 11). Although EBV-specific antibodypatterns reflect the dynamics of EBV activity in adults, few studies

have addressed this issue in infants and children (3, 1215) or innewborns (16, 17). More importantly, no comparison to maternalantibody levels has been made and there has been no analysis ofhow maternal malaria infections impact transplacental transfer ofEBV-specific antibodies.

Mothers transfer pathogen-specific antibodies to their infantsduring pregnancy. These passive antibodies from the mothersprovide protection to the infants before they develop de novo an-

Received 1 May 2015 Returned for modification 30 May 2015Accepted 12 September 2015

Accepted manuscript posted online 16 September 2015

Citation Ogolla S, Daud II, Asito AS, Sumba OP, Ouma C, Vulule J, Middeldorp JM,Dent AE, Mehta S, Rochford R. 2015. Reduced transplacental transfer of a subset ofEpstein-Barr virus-specific antibodies to neonates of mothers infected withPlasmodium falciparum malaria during pregnancy. Clin Vaccine Immunol22:11971205. doi:10.1128/CVI.00270-15.

Editor: M. F. Pasetti

Address correspondence to Rosemary Rochford,[email protected].

* Present address: Collins Ouma, Health Challenges and Systems, AfricanPopulation and Health Research Center, Nairobi, Kenya; Rosemary Rochford,Department of Immunology and Microbiology, University of Colorado, Denver,Aurora, Colorado, USA.

Copyright 2015, American Society for Microbiology. All Rights Reserved.

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tibodies (18). These antibodies are mainly acquired throughtransplacental transfer. The transport of maternally derived IgGacross the placenta is mediated by Fc receptors of IgG, includingFcR and FcRn (1921). It is an active and selective processwhereby neonatal FcR binds IgG, crosses the syncytiotrophoblast,and releases IgG into the endothelium of fetal capillaries.

Maternal factors such as placental malaria, human immuno-deficiency virus (HIV) infection, maternal hypergammaglobu-linemia, and preterm birth have been shown to inhibit the efficientmother-to-child transfer of pathogen-specific antibodies (2227).For example, in a study in the rural coastal area of Kenya, placentalmalaria infection as well as HIV infection was associated with asignificant reduction in the transfer of anti-tetanus IgG antibodiesto the neonates (26). In a rural Gambian population, placentalmalaria infection was associated with a significant reduction intransplacental transfer of antibodies against herpes simplex virus(HSV), varicella-zoster virus (VZV), and respiratory syncytial vi-rus (RSV) (27). Maternal HIV infection was associated with re-duced transplacental transfer of IgG antibodies against tetanustoxoid (TT), measles virus, and varicella-zoster virus (22). To-gether, these studies suggest that maternal infections during preg-nancy can interfere with the efficient vertical transfer of patho-gen-specific antibodies, potentially leaving infant susceptibleto infections early in life. Transplacental transfer of EBV-spe-cific maternal antibodies to their infants in the context of ma-ternal malaria infection has not been investigated.

Given that over 35% of infants from a region of malaria ende-micity can be infected with EBV during infancy (3), when mater-nally acquired antibodies should typically protect them againstEBV infection, we tested the hypothesis that maternal malariainfection reduced the transplacental transfer of EBV-specific ma-ternal antibodies to the neonates. In addition, the above-men-tioned studies looked at antibody transfer in the context of pla-cental malaria infection as determined by the presence of malariaparasites during delivery. The long-term effect of maternal ma-laria infection (e.g., infection with malaria at any time duringpregnancy) on antibody transfer remains poorly understood. Thisstudy investigated the effect of maternal malaria infection duringpregnancy on the efficiency of transplacental transfer of well-char-acterized EBV-specific antibodies (e.g., VCA, EBNA1, EAd, andZta) and tetanus toxoid-specific antibodies in mother-child pairsfrom a region of western Kenya where malaria transmission ishigh.

MATERIALS AND METHODSStudy population. This study was conducted at the antenatal clinic(ANC) and maternity ward of Chulaimbo Sub-District Hospital in Ki-sumu West District. This hospital mainly serves a rural population thatexperiences holoendemic malaria with two seasonal peaks: June to Augustand November to December (28). The inclusion criteria specified preg-nant women of any gravidity, gestation for 24 weeks, having a normalfull blood count, HIV-negative status, residency within a 10-km distanceof the hospital, and willingness to return to the hospital for delivery, fol-low-up clinical procedures, and laboratory testing. Gestational age wasevaluated by measurement of fundal height and history (the last men-strual period). All pregnant women were enrolled in a 6-month periodfrom June to November 2011. The demographics of this study populationhave been previously described (29). Briefly, at enrollment, demographicand antenatal care data were captured. A total of 200 pregnant womenwere enrolled. Of these, 25 were HIV positive and were excluded fromthese analyses. The remaining 175 pregnant women who were HIV-1

negative were followed up monthly through antenatal visits (up to 4 fol-low-up visits per mother) until delivery. Of these, 93 pregnant womendelivered at the hospital and 70 of them provided a complete mother-child pair of plasma samples that were analyzed in the current study.

All the women included in the study were tested for HIV as a part ofprograms investigating mother-to-child-transmission (MTCT) of HIV inaccordance with the Kenya Ministry of Health national guidelines. All themothers received an average of two doses of sulfadoxine-pyrimethamine(SP) that were administered by directly observed therapy during the ANCvisits as part of the recommendation of the Kenya Ministry of Health formalaria prophylaxis. Approvals for this study were obtained from theKenya Medical Research Institute (KEMRI) Ethical Review Committeeand the Ethical Review Board of the State University of New York (SUNY)Upstate Medical University Hospital, USA. Written informed consent wasobtained from all the mothers.

Sample collection and preservation. During the ANC visits andwithin 12 h of delivery, 200 to 500 l of maternal venous blood wascollected by venipuncture or finger prick and placed into EDTA Micro-tainers (BD, Franklin Lakes, NJ). After delivery, 500 l of cord blood,representative of the pool of blood circulating in the neonates, was col-lected from the umbilical vein into EDTA Microtainers (BD, FranklinLakes, NJ). All samples were immediately transported to the SUNY Up-state University laboratory at KEMRIs Centre for Global Health Re-search, and plasma was separated from whole blood and stored at 80Cuntil antibody assays were performed.

Malaria diagnosis. Malaria parasite load and plasmodium specieswere determined in maternal and cord blood samples as previously de-scribed (29). An infant was considered exposed to malaria in utero if any ofthe blood smear and/or quantitative (Q)-PCR results determined duringany of the ANC visits or any of the samples of maternal blood at deliveryor samples of cord blood were positive for P. falciparum. An infant wasconsidered not malaria exposed if the blood smear and/or Q-PCR resultsat any of the ANC visits or maternal blood samples at delivery or cordblood samples were negative.

EBV peptide antigens and Luminex-based suspension bead assays.EBV-specific antibodies were detected using 5 synthetic peptides coveringimmunodominant epitopes of the viral capsid antigen VCA-p18 subunit,VCA gp125 subunit, EBV nuclear antigen 1 (EBNA1), early diffuse anti-gen complex (EAd), and immediate-early protein Z transcriptional acti-vator (Zta) (13, 30, 31). EBV gp125 is another major capsid immunogenof VCA that is independent of p18. Tetanus toxoid (TT) antigen (Calbi-ochem, Darmstadt, Germany) was used as a control since mothers areroutinely immunized against tetanus during pregnancy. The choices ofthe above-mentioned antigens were based on their well-characterized se-rological profiles as previously described (13, 32, 33).

To detect EBV- and TT-specific IgG using the panel of EBV peptidesand TT described above, we used a Luminex-bead-based suspension assayand a previously described protocol (13). Plasma was diluted 1:100 priorto testing. At least 75 beads per region were acquired on a Bioplex reader(Bio-Rad, Hercules, CA), and results were expressed as mean fluorescenceintensity (MFI) values.

VCA- and EBNA1-specific IgG subclass ELISA. EBV-specific IgGsubclass antibody distributions were analyzed in the mother-child pairs ofplasma samples using two synthetic peptides covering the immunodom-inant epitopes of VCA-p18 and EBNA1 by enzyme-linked immunosor-bent assay (ELISA) as previously described (13, 32) with modifications.Peroxidase-conjugated sheep antibodies specific to the different humanIgG subclasses (Invitrogen, Camarillo, CA) were added and the platesdeveloped using tetramethylbenzidine (TMB) substrate. Plasma sampleswere diluted 1:100. The optical densities (ODs) were measured at 490 nmon a Bio-Rad microplate reader using Microplate Manager version 6 (Bio-Rad, Hercules, CA). Antibody levels for IgG subclass responses were ex-pressed in arbitrary units (AU), which were calculated by dividing the testsample ODs by the mean OD from EBV negative-control sera plus 2standard deviations (SDs).

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Total IgG ELISA. Determination of total IgG in maternal plasma wasdone using a human IgG total ELISA kit from eBioscience (San Diego,CA) following the manufacturers instructions. The plates (Molecular De-vices, Sunnyvale, CA) were read at 450 nm, and the concentration of totalIgG (in milligrams per milliliter) in plasma was extrapolated from thestandard curve.

Statistical analysis. The differences in the levels of EBV- and TT-specific IgG antibodies between malaria-exposed and nonexposed neo-nates as well as between malaria-infected and uninfected mothers weredetermined using the Mann-Whitney U test. Placental transfer was mea-sured as the ratio of the level of specific antibody in cord blood to that inthe maternal blood, i.e., the cord blood/maternal blood ratio (CMR) (26,27). The correlation between the levels of maternal EBV- and TT-specificantibodies in relation to those in cord blood was calculated using theSpearman correlation test. Linear regression analysis was used to assessthe effect of a number of variables such as maternal age, parity, birthweight (BW), and gestational age at first malaria exposure on the trans-placental transfer of EBV-specific antibodies. In multivariate analysis, weadjusted for maternal age, hypergammaglobulinemia, and gestational ageat exposure as potential confounders, as they are known or suspected riskfactors affecting outcomes. Statistical analyses were performed usingGraphPad Prism version 6 (GraphPad Software) and Stata statistical soft-ware version 11 (StataCorp). For all statistical analyses, a two-sided Pvalue of 0.05 was considered significant.

RESULTSCharacteristics of the study population. Plasma from 70 mother-child pairs was analyzed in this study. A neonate was consideredexposed to malaria if P. falciparum malaria was detected in mater-nal venous blood at any ANC visit, in the placenta or venous bloodat delivery, or in cord blood. A neonate was considered nonex-posed if P. falciparum malaria was not detected in the maternalvenous blood at any ANC visit, at delivery, or in cord blood. It ispossible that we did not capture all malaria infections in themother, but for the purpose of this comparison, we labeled thisgroup nonexposed. Using this criterion, 63% (44/70) of the neo-nates in this study were classified as having been exposed to ma-laria in utero (Table 1).

The demographic and clinical characteristics of this study pop-ulation are shown in Table 1. The mean maternal age in the ma-laria-exposed group was 22.36 years (SD, 6.1), whereas that inthe nonexposed group was 21.77 years (SD, 6.79). To determineif the mothers were infected with EBV, anti-VCA-p18 IgG anti-body levels were measured in the enrollment ANC plasma samplesby ELISA (13, 32). All the women in this study were EBV seropos-itive. A total of 95% of the pregnant mothers in the malaria-ex-posed group and 92% in the malaria-nonexposed group had re-ceived tetanus toxoid (TT) vaccine during the current pregnancy.The proportions of primigravid mothers in the malaria-exposedgroup and in the nonexposed group were 36% and 30%, respec-tively. Only four neonates weighed less than 2.5 kg, which is de-fined as representing low birth weight (LBW). There were 2 (5%)infants with LBW in the malaria-exposed group and 2 (8%) in themalaria-nonexposed group. Hypergammaglobulinemia was de-fined as representing levels of total IgG greater than 30 mg/ml.Seven (27%) mothers in the malaria-uninfected group and 16(36%) mothers in the malaria-infected group had hypergam-maglobulinemia.

Comparison of levels of EBV- and TT-specific antibodies inmothers and their neonates. We first compared the levels of EBV-and TT-specific antibodies in mothers and their paired neonatesto determine if malaria exposure during pregnancy affected the

levels of antibodies in the mothers or in the cord blood of theinfants. To do this, we used a Luminex-bead-based assay that al-lowed us to measure antibodies to EBV lytic (e.g., VCA-p18, VCA-gp125, EAd, and Zta) and latent (e.g., EBNA1) antigens as well asTT antigen (13). We included the TT antigen as a reference anti-gen, as there are numerous studies evaluating the transplacentaltransfer of anti-TT antibody (22, 26, 34, 35). The levels of EBV-specific anti-VCA-p18, anti-VCA-gp125, and anti-TT antibodieswere comparable in the mothers and their neonates irrespective ofmalaria exposure status (Fig. 1). In contrast, both the malaria-exposed and malaria-nonexposed neonates had significantlylower levels of anti-Zta and anti-EAd antibodies in the cord bloodthan were seen in their mothers (both P 0.001). Interestingly,malaria-exposed neonates had significantly lower levels of anti-EBNA1 antibodies than were seen in their mothers (P 0.001),whereas there was no significant difference in the levels of anti-EBNA1 antibodies between the mothers and their neonates thatwere not exposed to malaria (Fig. 1). Because the pregnant moth-ers were infected with malaria at different time points duringpregnancy, we compared the levels of anti-EBV and anti-TT anti-bodies in neonates who were exposed to malaria early (26 weeksgestation) versus late (26 weeks gestation) in pregnancy andfound no significant difference in the levels of anti-VCA-p18,anti-EBNA1, anti-Zta, anti-EAd, anti-VCA-gp125, and anti-TTbetween the two groups of neonates (data not shown).

Since we observed similar levels of anti-VCA-p18, anti-VCA-gp125, and anti-TT in the mothers and their neonates, we thencorrelated the levels of anti-EBV- and anti-TT-specific antibodiesin maternal venous blood to the levels in their neonates to deter-

TABLE 1 General characteristics of the mothers and their infantsa

Factor

Values for indicated subjectmalaria exposure condition

P valueExposed Unexposed

Maternal characteristicsn 44 (63) 26 (37) 0.041Age, yr (mean) [SD] 22.36 [ 6.10] 21.77 [ 6.79] 0.566

20 yr 18 (41) 12 (46) 0.79020 yr 26 (59) 14 (54) 0.763

Gestational age37 wks 8 (18) 4 (15) 0.90037 wks 33 (75) 18 (69) 0.648

GravidaPrimigravida 16 (36) 7 (30) 0.784Secundigravida 12 (28) 12 (40) 0.548Multigravida 16 (36) 7 (30) 0.784

TT vaccine (currentpregnancy)

42 (95) 24 (92) 0.627

Bed net use 32 (73) 20 (77) 0.750EBV seroprevalence (%) 44 (100) 26 (100) 1.000Mean total IgG [SD] (mg/ml) 29.15 [ 10.12] 25.29 [ 8.93] 0.112

Neonate characteristicsMean birth wt (g) [SD] 3,171 [ 444] 3,146 [ 420] 0.923Time of malaria exposure

Early 31 (70)Late 13 (30)

a Data are presented as number (percent) unless otherwise stated. Differences inproportions between the groups were determined by the Fisher exact test. Earlyexposure to malaria was defined as 26 weeks gestation; late exposure was defined as26 weeks gestation.

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mine if the maternal antibody level is a predictor of neonatal an-tibody levels. We observed a significant positive correlation be-tween levels of maternal and neonatal anti-VCA-p18, anti-EBNA1, anti-gp125, and anti-TT antibodies regardless of malariaexposure (Fig. 2). However, there was no significant correlation inthe levels of anti-Zta and anti-EAd antibodies in the malaria-non-exposed group.

Neonates born to mothers with malaria during pregnancyhave reduced transplacental transfer of anti-VCA-p18 and anti-EBNA1 antibodies. We next determined if there was a reductionin EBV-specific antibody transfer due to exposure to malaria inutero. Placental transfer was measured as the ratio of the level ofantibody in cord blood to that in the maternal venous blood atdelivery, i.e., the cord blood/maternal blood ratio (CMR). Trans-

placental transfer of antibodies to VCA-p18 and EBNA1 frommalaria-infected mothers to their exposed neonates was signifi-cantly reduced (by 13.40% and 21.65%, respectively) (Table 2).There was no significant reduction in transplacental transfer ofanti-Zta, anti-EAd, and anti-TT from the mothers to the neonatesdue to malaria exposure in utero (P 0.950, P 0.349, and P 0.458, respectively), although there was a trend to a more signifi-cant reduction of anti-VCA gp125 (P 0.064) (Table 2).

When we performed a multivariate analysis to determine theeffect of maternal malaria infection on transplacental transfer ofanti-EBV and anti-TT antibodies while adjusting for potentialconfounding factors such as maternal age, hypergammaglobu-linemia status, and gestational age at first malaria exposure, weobserved that transfer of anti-VCA-p18 and anti-EBNA1 antibod-

FIG 1 Relative levels of EBV- and TT-specific IgG antibodies in malaria-exposed and nonexposed mothers and infants. Plasma was diluted at 1:100 and testedusing a Luminex-bead-based assay. The mean fluorescence intensity (MFI) of 75 Luminex beads for each of the antigens tested is indicated on the y axis.Significant P values of paired t tests are indicated in the figures. Horizontal bars represent median values.

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ies to the neonates from mothers who had malaria infection dur-ing pregnancy was significantly lower than that seen with thosewho did not have malaria during pregnancy (P 0.009 and P 0.042, respectively) (Table 3). No significant reductions were ob-served for the other EBV-specific antibodies or for TT-specificantibodies.

VCA-p18- and EBNA1-specific IgG subclass distributions inmaternal venous blood and cord blood. One possible explana-tion for the reduced transplacental transfer of anti-VCA and anti-EBNA antibodies could be differences in the IgG subclass distri-

butions of these antibodies during pregnancy, potentially due toviral reactivation. To test this possibility, we assessed IgG subclassreactivity to EBNA1 and VCAp18 antigens in the maternal venousblood and in the infant cord blood. Anti-VCA-p18 IgG1 and IgG4were detected in all the maternal and cord blood samples. A totalof 42% and 40% of the cord blood samples had detectable anti-VCA-p18 IgG2 and IgG3 antibodies, respectively. EBNA1 IgG1was detected in 98% and 88% of the maternal and cord bloodsamples, respectively. A total of 83% of maternal and cord bloodsamples had detectable EBNA1 IgG2, while EBNA1 IgG3 was de-

FIG 2 Correlation between maternal and infant anti-EBV- and anti-TT-specific antibodies in malaria (Mal)-exposed and nonexposed groups. The correlationbetween maternal and infant antibody levels was assessed by Spearman correlation, where P 0.05 was considered significant, and only significant P values areshown in the figure.




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tected in 71% and 59% of the maternal and cord blood samples. Atotal of 35% and 36% of maternal blood and cord blood haddetectable EBNA1 IgG4, respectively.

Next, we evaluated the levels of anti-VCAp18 and anti-EBNA1IgG subclass levels in maternal venous blood and cord blood sam-ples. Levels of VCA-p18- and EBNA1-specific IgG1 were signifi-cantly lower in cord blood than in maternal venous blood (P 0.001 and 0.012, respectively). There were comparable levels ofVCA-p18-specific IgG2, IgG3, and IgG4 in maternal venous bloodand cord blood. Similarly, we did not observe any significant dif-ference in the levels of EBNA1-specific IgG2, IgG3, and IgG4 be-tween maternal venous blood and cord blood (all P 0.05) (Fig.3). There were higher overall levels of anti-EBNA1 IgG2 antibod-ies than of the other IgG isotypes.

DISCUSSION

This study tested the hypothesis that exposure to P. falciparummalaria in utero would interfere with the transplacental transfer ofEBV-specific antibodies from the mother to the neonate. Indeed,the data presented in this study demonstrate that transfer of IgGantibodies to the EBV lytic VCA-p18 antigen and latent EBNA1antigen from the mother to the neonate was significantly reducedfollowing malaria exposure in utero and that this effect remainedsignificant even after adjusting for other confounding factors. Wealso observed that, regardless of malaria exposure during preg-nancy, neonates had significantly lower levels of EBV lytic anti-Ztaand anti-EAd IgG antibodies.

A number of conditions are known to affect the maternal-fetaltransfer of IgG antibodies, including HIV infection, placental ma-laria, and maternal hypergammaglobulinemia (23, 26, 27, 34). Inthis study, only HIV-negative mothers were enrolled, and we ad-justed for hypergammaglobulinemia in our multivariate analysis,

with results indicating that the effect of reduced transfer of anti-VCA-p18 and anti-EBNA1 antibodies was likely due to maternalmalaria infection during pregnancy. It is not clear why this effect isselective for only two of the EBV antigens tested. However, wehave previously shown that malaria infection during pregnancyresults in EBV reactivation (29). It is possible that there was a shiftin antibody isotype responses during EBV reactivation in themothers such that a different isotype of IgG was dominant and wasless efficiently transferred to the neonates. It is known that anti-gens that mainly elicit IgG1 or IgG3 responses are transportedacross the placenta more efficiently than either IgG2 or IgG4 (36,37), with a consequence of reduced transplacental transfer of an-tibodies of these specificities. A previous study found that the IgGsubclass distribution to VCA in acutely infected individuals orduring viral reactivation was predominantly IgG1, no IgG2 wasdetected, and the presence of IgG3 was indicative of viral reacti-vation (12). Consistent with that study, we found that IgG1 wasthe dominant subclass of IgG in anti-VCA and anti-EBNA anti-bodies detected in maternal venous blood and infant cord blood.Thus, the reduction in transplacental transfer was likely due todecreased IgG1 transfer and not to a shift in IgG subclasses.

Antibodies to the EBV EAd lytic antigen have long been usedclinically as a marker of EBV reactivation, as they are short-lived(38). More recently, studies conducted with another EBV lyticantigen, Zta, have also shown utility in indicating viral reactiva-tion (10, 13). All the mothers in this study had both anti-Zta andanti-EAd antibodies at the time of delivery indicative of viral re-activation. This is consistent with studies that have demonstratedthat pregnancy induced viral reactivation (39, 40). One surprisingfinding from this study is that, irrespective of malaria exposure,antibodies against Zta and EAd are not efficiently transportedacross the placenta into fetal circulation, resulting in low levels of

TABLE 2 Transplacental transfer of EBV- and TT-specific antibodies from the mother to the neonatea

Antibody

CMR for indicated subject malaria exposure condition

% reduction P valueExposed Nonexposed

Anti-VCA-p18 0.862 (0.7541.001) 0.996 (0.8291.182) 13.40 0.023Anti-EBNA1 0.546 (0.4260.809) 0.762 (0.5581.012) 21.65 0.002Anti-Zta 0.151 (0.0800.313) 0.153 (0.0740.320) 0.25 0.950Anti-EAd 0.102 (0.0480.176) 0.068 (0.0350.166) 3.40 0.349Anti-VCA-gp125 0.697 (0.5520.979) 0.850 (0.6800.972) 15.25 0.064Anti-TT 0.949 (0.8160.103) 0.981 (0.8541.040) 3.20 0.458a Data represent the median CMR (cord blood/maternal blood ratio; i.e., ratio of the level of the specific antibody in cord blood to that in the maternal blood of the mother).Interquartile ranges are shown in parentheses. Maternal and cord blood plasma samples were tested for the presence of anti-EBV- and anti-TT-specific antibodies by Luminex-bead-based assay. The CMR was used to determine levels of placental transfer of antibodies. Percentage reduction due to malaria was determined by the following formula: (CMRof nonexposed subject CMR of exposed subject) 100. Statistical differences of P 0.05 are considered significant as determined by the Mann-Whitney test. Boldface dataindicate a significant difference in the transfer of EBV-specific antibodies between the two groups.

TABLE 3 Multivariate linear regression analysis of the transplacental transfer of EBV- and TT-specific antibodiesa

Outcome Intercept SE Mean difference SE P value

Log anti-VCA-p18 0.014 0.330 0.573 0.212 0.009Log anti-EBNA1 0.703 0.395 0.526 0.253 0.042Log anti-Zta 2.065 0.749 0.644 0.480 0.185Log anti-EAd 2.681 0.998 0.466 0.640 0.469Log anti-VCA-gp125 0.029 0.367 0.215 0.235 0.365Log anti-TT 0.213 0.270 0.143 0.173 0.410a The CMR data were log transformed, and then values were adjusted for neonatal values plus maternal age, hypergammaglobulinemia status, and gestational age at exposure.Boldface data indicate a significant association between maternal malaria infection during pregnancy and the transfer of the EBV-specific antibodies.

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FIG 3 Levels of EBV-specific IgG subclass in venous blood of mothers and their neonates. Anti-VCA-p18-specific (A) and anti-EBNA1-specific (B) IgG subclassantibodies in cord blood (CB) and maternal venous blood (VB) at delivery were measured by ELISA. Arbitrary units were calculated by dividing the test sample ODs bythe mean OD plus 2 standard deviations (SDs) of the negative-control sera. Horizontal bars represent the means, and only significant P values are shown in the figure.




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anti-Zta and anti-EAd antibodies in the neonates. One possibleexplanation is the low maternal levels of anti-Zta and anti-EAdantibodies. This observation is supported by data from a previousstudy that demonstrated that levels of maternal antibodies to vari-cella-zoster virus represented the most important predictor of theneonatal antibody levels (41). The low maternal levels of anti-EAdantibodies could have been due to the fact that the levels of anti-EAd antibodies fall relatively quickly after infection (42). Since themajority of IgG is transported across the placenta during the lasttrimester of pregnancy (43), the rapid decay of anti-EAd antibod-ies may result in very few antibodies being transferred to the neo-nates, as reported. This is in contrast to the high levels of IgGantibodies against both VCA-p18 and EBNA1 that we observedand that persist for life in healthy carriers (42). This could alsoexplain the lack of correlation between maternal and neonatalanti-EAd antibodies observed in the current study. Since someneonates in both the malaria-exposed and nonexposed groupshad elevated anti-EAd IgG levels suggestive of active neonatal in-fection, EBV DNA was assessed in all the cord blood samples, butthe results were found to be negative (data not shown).

Another plausible explanation for the low levels of anti-Zta andanti-EAd IgG antibodies in cord blood is the competitive bindingof antibodies to the neonatal Fc receptors. The transfer of antibod-ies across the placenta is a selective process that is dependent onneonatal Fc receptors (FcRn). For antibodies to be transferredacross the placenta, they have to bind to the FcRn and be trans-ported across the syncytiotrophoblast and into the fetal circula-tion (20, 43). Antibodies that are expressed at high levels in themother may be competing with the antibodies that are expressedat low levels, such as anti-Zta and anti-EAd, for binding to thelimited number of FcRn receptors, thereby reducing the transferof anti-Zta and anti-EAd antibodies to the infants. However, thisobservation warrants further studies to investigate the mecha-nism. Lastly, the low levels of cord blood anti-Zta and anti-EAdantibodies could be explained by the fact that these antibodies mayhave been bound to the specific antigen and trapped within theplacenta as immune complexes (44) and, as a consequence, nottransported into the fetal circulation.

The finding from this study that maternal malaria infectionduring pregnancy did not affect the transplacental transfer of TT-specific antibodies is consistent with previous studies that demon-strated that placental malaria does not have an effect on the trans-placental transfer of anti-TT antibodies (22, 35). However, theseresults are in contrast to other studies that demonstrated that placen-tal malaria results in reduced transplacental transfer of anti-TT anti-bodies (26, 34). However, all of these previous studies focused onplacental malaria as determined by the presence of malaria parasitesin the mother at birth. In contrast, we assessed the presence of malariainfection at any time during pregnancy; thus, our results are not di-rectly comparable with the results from previous studies.

A strength of this study was the longitudinal study design,which allowed us to actively follow pregnant mothers throughpregnancy up to delivery, whereas other studies have focusedmainly on placental malaria due to their cross-sectional study de-sign. Our approach allowed us to identify peripheral malaria in-fections of the mother during pregnancy rather than only malariainfection at delivery. In addition, we used a Q-PCR method thatwas more sensitive (compared to methods employing bloodsmears) to determine the malaria infection status of the pregnantmothers at enrollment, at subsequent follow-up, and at delivery

and the malaria infection status of cord blood. However, whetherthe mothers had single or repetitive infections with malaria wasnot analyzed, which represents a limitation of the study. We didnot analyze mothers with placental malaria separately from thosewith malaria infection at any time during pregnancy because therewere so few mothers with placental malaria in this cohort. Anadditional limitation of our study was the modest number ofmother-child pairs included in these analyses. Finally, while anti-VCA, anti-EBNA1, anti-EAd, and anti-Zta antibodies are well de-scribed during infection with EBV, they are not known to be neu-tralizing antibodies. In contrast, antibodies against the EBV gp350protein are neutralizing but have not been well characterized inhuman populations. Future studies will need to be done to analyzethe levels of anti-gp350 antibodies in this cohort.

In summary, malaria infection during pregnancy results in im-paired transplacental transfer of a subset of EBV-specific antibod-ies. Inadequate transfer of anti-EBV antibodies from mothers tothe neonates may predispose the infants to early EBV infection.Infection with EBV in infancy leads to poor control of the virusand has been hypothesized to be a risk factor for Burkitts lym-phoma. It remains to be determined whether this reduced trans-placental transfer of anti-EBV antibodies predisposes the neo-nates to EBV infection in early infancy. This report demonstratesthe need for improved measures to prevent maternal malaria in-fections during pregnancy.

ACKNOWLEDGMENTS

We acknowledge the Chulaimbo Sub-District Hospital for allowing us touse their facilities to perform this study. We also thank our clinical officersand data entry and field staff members involved in the project and M. M.Majiwa for his invaluable insights. We are particularly grateful to themothers and their infants for participating in this study.

The manuscript was submitted for publication with the permission ofthe Director of KEMRI.

We do not have any commercial or other association that might posea conflict of interest.

Funding was provided through the National Cancer Institute at theNational Institutes of Health (CA102667 [R.R.]). S.O. is a Ph.D. ResearchFellow supported by a D43 training grant (NCI 153707). The funders hadno role in study design, in data collection and interpretation, or in thedecision to submit the manuscript for publication.

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Reduced Transplacental Transfer of a Subset of Epstein-Barr Virus-Specific Antibodies to Neonates of Mothers Infected with Plasmodium falciparum Malaria during PregnancyMATERIALS AND METHODSStudy population.Sample collection and preservation.Malaria diagnosis.EBV peptide antigens and Luminex-based suspension bead assays.VCA- and EBNA1-specific IgG subclass ELISA.Total IgG ELISA.Statistical analysis.

RESULTSCharacteristics of the study population.Comparison of levels of EBV- and TT-specific antibodies in mothers and their neonates.Neonates born to mothers with malaria during pregnancy have reduced transplacental transfer of anti-VCA-p18 and anti-EBNA1 antibodies.VCA-p18- and EBNA1-specific IgG subclass distributions in maternal venous blood and cord blood.

DISCUSSIONACKNOWLEDGMENTSREFERENCES

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